Regulatory mechanisms controlling synaptogenesis are key to the normal development of the CNS. Much of our knowledge of synaptogenesis has been gained from the neuromuscular junction. This information has now permitted us to formulate hypotheses about the regulatory mechanisms controlling the formation of synaptic circuitry of mammalian central neurons. Using rat hippocampal neurons in cell culture we will elucidate regulatory mechanisms controlling the development of mammalian presynaptic nerve terminals. Knowledge gained in this study will be crucial for developing strategies to restore or repair damaged circuitry in the CNS. Specific Aim I: The developmental appearance of functional presynaptic calcium channels: The relative roles of intrinsic and extrinsic regulation. Using calcium imaging and patch clamp techniques on cell cultured hippocampal neurons, we will determine the developmental timetable for expression of functional calcium channels. We will determine effects of neuron-neuron contact on the expression of functional presynaptic calcium channels responsible for neurotransmitter release. Specific Aim II The role of glial-neuronal signaling in synaptogenesis. Electrophysiological evidence from our laboratory indicates that astrocytes regulate neuronal synapse development. We will ask which aspects of presynaptic development are regulated by astrocytes? Specific Aim III Is the rate-limiting step in synaptogenesis the expression of functional calcium channels or of functional secretory apparatus? By performing electrophysiology together with calcium imaging and flash photolysis of caged calcium (DM-Nitrophen) we will determine whether the rate-limiting step in synaptogenesis is due to the timing of expression of functional calcium channels or of functional calcium- regulated secretory apparatus. Specific Aim IV Signal transduction pathways controlling presynaptic development. Working from evidence obtained in peripheral synapses, we will now test the roles of cAMP and internal calcium, as well as other second messengers, as regulators of synaptogenesis of mammalian central neurons. This investigation will represent one of the first systematic cellular studies of synaptogenesis between mammalian central neurons. Using approaches that have been previously shown to be successful for peripheral neurons, we will obtain some of our first insights into the cell and molecular regulatory mechanisms which control the development of central presynaptic terminals.